Low detection of microRNAs that are closely related to the specific target. Amplification plots and melting curves for amplification of specific targets (ssc-let-7a, ssc-miR-125b and mmu-miR-200b-3p) and closely related miRs with one base mismatch to the forward primer (ssc-let-7e), to the reverse primer (ssc-miR-125c) and to each of the primers (mmu-miR-200c-3p). The position of the mismatch is indicated with a box on the alignment of primers and microRNAs. Only the microRNA-specific bases of the reverse primers are shown in the alignment. The curves labeled “ntc” are non-template controls. The experiment was performed as previously described using the same primers for ssc-let-7a and ssc-miR-125b . QPCR of mmu-miR-200c-3p was done with the Brilliant III Ultra-Fast QPCR Master Mix (Agilent, USA). The sequence of the primers can be found in Additional file 5 (ssc-miR-125b is identical to mmu-miR-125b).